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Am J Physiol Heart Circ Physiol 284: H1182-H1189, 2003. First published December 19, 2002; doi:10.1152/ajpheart.00680.2002
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Vol. 284, Issue 4, H1182-H1189, April 2003

Increased myofibrillar protein phosphatase-1 activity impairs rat aortic smooth muscle activation after hypoxia

Hwee Teoh, Mary Zacour, Avraham D. Wener, Lakshman Gunaratnam, and Michael E. Ward

Terrence Donnelly Laboratories, Division of Respirology and Department of Critical Care, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada M5B 1W8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that increased myofibrillar type 1 protein phosphatase (PP1) catalytic activity contributes to impaired aortic smooth muscle contraction after hypoxia. Our results show that inhibition of PP1 activity with microcystin-LR (50 nmol/l) or okadaic acid (100 nmol/l) increased phenylephrine- and KCl-induced contraction to a greater extent in aortic rings from rats exposed to hypoxia (10% O2) for 48 h than in rings from normoxic animals. PP1 inhibition also restored the level of phosphorylation of the 20-kDa myosin light chain (LC20) during maximal phenylephrine-induced contraction to that observed in the normoxic control group. Myofibrillar PP1 activity was greater in aortas from rats exposed to hypoxia than in normoxic rats (P < 0.05). Levels of the protein myosin phosphatase-targeting subunit 1 (MYPT1) that mediates myofibrillar localization of PP1 activity were increased in aortas from hypoxic rats (193 ± 28% of the normoxic control value, P < 0.05) and in human aortic smooth muscle cells after hypoxic (1% O2) incubation (182 ± 18% of the normoxic control value, P < 0.05). Aortic levels of myosin light chain kinase were similar in normoxic and hypoxic groups. In conclusion, after hypoxia, increased MYPT1 protein and myofibrillar PP1 activity impair aortic vasoreactivity through enhanced dephosphorylation of LC20.

smooth muscle contraction; myosin light chain kinase; myosin light chain phosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DURING ACUTE REDUCTIONS in oxygen delivery, vital organ oxygenation is preserved by sympathetically mediated vascular reflexes that redirect blood flow (21) and enhance oxygen extraction (7). If hypoxia is prolonged (12-48 h), however, the reactivity of systemic arteries (2, 32) and sympathetic responses (12) are impaired. This will limit the ability to maintain vital organ oxygen supply if substrate supply must be increased to meet an increase in metabolic demand in the event of superimposed hypotension or if hypoxia acutely becomes more severe. Despite its clinical and physiological relevance, the mechanisms that mediate the effect of hypoxia on systemic vascular smooth muscle contractility are unknown.

Smooth muscle contraction is regulated by phosphorylation of the 20-kDa myosin light chain (LC20) (17, 18) by myosin light chain kinase (MLCK) and the opposing action of myosin light chain phosphatase (MLCP). MLCP is a type 1 protein phosphatase (PP1) consisting of a catalytic (38 kDa) and two noncatalytic (110-130 kDa and 20 kDa) subunits (1, 8). The large noncatalytic subunit, myosin phosphatase targeting subunit 1 (MYPT1), is responsible for localization of phosphatase activity to the myosin heavy chain and is the site of phosphorylation-dependent regulation (5, 19). Increased myofibrillar PP1 activity contributes to cGMP-mediated vasorelaxation (22), whereas inhibition of this activity is an important mechanism by which contraction is enhanced during alpha -adrenoceptor stimulation (20) and has been implicated in the pathophysiology of arterial vasospasm (10). Recently, we (37) reported that LC20 phosphorylation during phenylephrine-induced contraction is reduced in aortic strips from rats exposed to hypoxia for 48 h compared with strips from normoxic animals, and an imbalance in the relative activities of MLCP and MLCK was proposed as a contributing factor in the contractile abnormality. Given this observation and the important role that changes in MLCP activity play in the short- and long-term regulation of vascular tone, we hypothesized that increased myosin-targeted PP1 activity may contribute to hypoxia-induced aortic hyporeactivity through enhanced dephosphorylation of LC20.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure to Hypoxia

In vivo studies. All animal procedures were approved by the institutional animal care committee. As described previously (2), male Sprague-Dawley rats (200-250 g) were placed in a Plexiglas chamber (30 cm × 18 cm × 15 cm), into which the flow of air and nitrogen was controlled independently, and from which gas outflow was through an underwater seal. Animals exposed to hypoxia breathed a mixture containing 10% O2, whereas control animals breathed air only under otherwise identical conditions. The thoracic aortas were excised immediately after decapitation and mounted in organ bath myographs containing Krebs-Henseleit solution (KHS) composed of (in mmol/l) 120 NaCl, 25 NaHCO3, 11.1 glucose, 4.76 KCl, 1.18 MgSO4, 1.18 KH2PO4, 2.5 CaCl2 aerated with 95% O2-5% CO2 at 37°C, or frozen in liquid nitrogen for later measurement of MYPT-1 protein levels.

Cell culture studies. Levels of MYPT-1 protein were also measured in human aortic smooth muscle cells (HASMCs) to assess whether the effect of hypoxia is independent of neurohumoral and hemodynamic changes and whether this effect is relevant to human vascular tissues. HASMCs (Clonetics) at passage 6 were grown to 80% confluence and incubated for 16 h or 48 h at 37°C under either normoxic (21% O2-5% CO2-74% N2) or hypoxic (1% O2-5% CO2-94% N2) conditions. Oxygen concentrations equal to or lower than that used in these studies have been recorded in the arterial wall in animals in vivo (4).

Contractile Responses

Rat aortas were bisected and one-half denuded of its endothelium by gentle abrasion of the luminal surface before being sectioned into 4-mm rings. Segments were equilibrated in warmed aerated KHS for 1 h under a resting tension of 2 g before drug-induced changes in tension were monitored. The failure of acetylcholine (1 µmol/l) to elicit relaxation after contraction with phenylephrine (1 µmol/l) was taken as evidence of functional endothelial ablation (2). A ring from each rat was incubated in KHS with or without vehicle (0.05% DMSO), okadaic acid (ODA; 1 or 100 nmol/l) or microcystin-LR (M-LR; 0.5 or 50 nmol/l) for 20 min before and during generation of concentration response curves to phenylephrine (0.1 nmol/l-100 µmol/l). To determine the effects of PP inhibition on the response to smooth muscle membrane depolarization, KCl concentration-response relationships (0-100 mmol/l) were generated in the presence and absence of either DMSO (0.05%) or M-LR (0.5 or 50 nmol/l) with the use of separate groups of aortic rings from normoxic and hypoxia-exposed rats. Two concentrations of ODA and M-LR were used to differentiate between PP1- and PP type 2A (PP2A)-mediated events as the former are less susceptible to these toxins than are the latter; hence the effects seen at the higher but not the lower concentrations are attributed to PP1 inhibition (8). On completion of the experiments, the rings were dried overnight at 50°C, and weighed to express tension as grams per milligram dry weight.

LC20 Phosphorylation

Each endothelium-denuded rat aorta was cut into three helical strips that were equilibrated for 1 h in warmed aerated KHS under 2 g of resting tension before being incubated for 20 min with 0.05% DMSO or 50 nmol/l M-LR and contracted with phenylephrine (10 µmol/l). LC20 phosphorylation was measured as described previously (37). A strip from each rat was freeze-clamped in liquid nitrogen at 0, 1, or 10 min after the addition of phenylephrine. Tissues were denatured in a frozen slurry of dry ice, liquid nitrogen, and 20 mmol/l DTT in 10% TCA-acetone before being repeatedly washed with DTT-free 10% TCA-acetone. LC20 protein was extracted from lyophilized samples in solution composed of 8 mol/l urea, 0.2 mol/l Tris, 0.22 mol/l glycine, 0.01 mol/l DTT, 0.6 mol/l KI, 0.1% bromophenol blue, 0.01 mol/l EGTA, and 0.001 mol/l EDTA for 3 h at room temperature. Constant volumes of these samples were electrophoresed (6 mA per gel) on freshly prepared nondenaturing gels [separating gel: 10%/0.27% acrylamide/bisacrylamide, 40% glycerol, 0.5% ammonium persulfate, 0.044% N,N,N',N-tetramethylethylenediamine (TEMED); stacking gel: 30%/1.6% acrylamide/bisacrylamide, 8.5 mol/l urea, 0.6% ammonium persulfate, 0.2% TEMED] in a running buffer of 0.05 mol/l Tris-0.1 mol/l glycine. Proteins were electrotransferred to nitrocellulose in 0.1 mol/l CAPS (pH 11) for 16 h at 4°C. Membranes were blocked in 5% milk-0.1% Tween-Tris-buffered saline (TBS), immunostained with a monoclonal LC20-specific antibody (1:1,000; Sigma) and detected with horseradish peroxidase (HRP)-goat anti-mouse IgG (1:1,000; Sigma). Immunocomplexes were detected by enhanced chemiluminescence (Amersham). A standard curve of chicken gizzard myosin (Sigma), extracted simultaneously with the experimental samples, was routinely loaded on the same gel to provide a positive control and a measure of linearity of the optical density curve. Tissues from control and hypoxic rats were always paired for extraction, electrophoresis, and immunoblotting. The extent of LC20 phosphorylation in each sample was quantitated by densitometry with the use of a Hewlett-Packard scanner and Molecular Analyst software (Bio-Rad Laboratories) before expression as the percentage of phosphorylated to total LC20 in that lane.

PP Activity

Cytosolic and myofibrillar extracts were prepared from whole rat thoracic aortas (10). Four aortas were pooled for each sample and the results from each sample taken as single values for statistical analysis. Samples were ground in liquid nitrogen and mixed with 500 µl of extraction solution (0.004 mol/l EDTA, 0.1% beta -mercaptoethanol, 0.001 mol/l benzamidine, and 0.0001 mol/l PMSF, pH 7.0). The suspension was centrifuged and the supernatant (first cytosolic extract) removed. The resultant pellet was blended with 500 µl of the cold extraction buffer and centrifuged to yield a second supernatant (second cytosolic extract). The pellet was homogenized with 500 µl of solution A, which was composed of 0.02 mol/l triethanolamine/HCl (pH 7.5, 4°C), 0.1% beta -mercaptoethanol, 0.001 mol/l benzamidine, and 0.0001 mol/l PMSF that contained 0.002 mol/l EGTA, 0.5% Triton X-100, and 0.0006 mol/l NaCl. After standing for 30 min at room temperature, this homogenate was diluted with an equal volume of solution A before being centrifuged. The resulting supernatant (myofibrillar extract) and both cytosolic extracts were then assayed for protein content (23). PP activities were determined by measuring the rate of liberation of radioactivity from [32P]phosphorylase by using a commercially available kit (GIBCO-BRL Life Technologies). PP1 activity was evaluated as PP activity in the presence of 0.5 nmol/l M-LR, whereas PP2A activity was assessed as PP activity inhibited by 0.5 nmol/l M-LR.

Western Blots

Whole thoracic aortas from rats exposed to normoxia or to hypoxia for 16 h and for 48 h were divided into two parts; one-half was denuded of endothelium by gentle abrasion. Aortic proteins were extracted in 1% SDS, 0.001 mol/l sodium orthovanadate, and 0.01 mol/l Tris (pH 7.4). Normoxia- and hypoxia-exposed HASMC were treated with 0.05 mol/l Tris (pH 8.0), 0.15 mol/l NaCl, 0.1% SDS, 0.001 mol/l PMSF, 1 mg/ml aprotinin, 1 mg/ml Nonidet P-40, and 0.5% sodium deoxycholate. The resulting rat aortic and HASMC lysates were resolved by SDS-PAGE (4-12%), electrotransferred to nitrocellulose, blocked in 5% milk-Tween-TBS, and incubated with antiserum specific for MYPT1 (1:300; Zymed). Proteins extracted from endothelium-intact aortas from rats exposed to normoxia or hypoxia for 48 h were electrophoresed and electrotransferred in the same manner but the membranes were incubated with antiserum specific for MLCK (1:5,000; Sigma). In all cases, protein concentration was determined by Lowry assay, and appropriate volumes of extraction buffer to produce constant protein loading in each lane were mixed with SDS loading buffer. Samples from normoxic and hypoxic groups were paired on each gel to control for interexperimental variation. Protein loading and transfer efficiency were verified after transfer with the use of full-lane densitometry of the Ponceau red-stained membranes. Immunoblots were probed with HRP-donkey anti-rabbit IgG (1:1,000; Amersham) and visualized by enhanced chemiluminescence (Amersham). Band intensity was quantified by densitometry using a Hewlett-Packard scanner and Molecular Analyst software (Bio-Rad Laboratories).

Data Analysis

Unless otherwise stated, results are presented as means ± SE; n refers to the number of samples, with P < 0.05 representing statistical significance. Paired means were compared by two-tailed Student's t-test. Differences among multiple means were evaluated by ANOVA, and when overall differences were detected, individual means were compared post hoc with the use of Bonferroni's test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Contractile Responses

The effects of ODA (1 and 100 nmol/l) and M-LR (0.5 and 50 nmol/l) on the responses to phenylephrine in rings from normoxic and hypoxia-exposed rats are illustrated in Fig. 1. Table 1 presents the maximum tensions and EC50 values during phenylephrine-induced contraction in the presence and absence of ODA and M-LR and the effect of M-LR on these values during KCl-induced contraction in aortic rings from normoxic and hypoxia-exposed rats. As in previous studies (2, 37), the maximum contractions elicited by phenylephrine and KCl in rings from normoxic rats were greater than those in rings from rats exposed to hypoxia. The EC50 values did not differ between the two groups. Concentrations of ODA (1 nmol/l) and M-LR (0.5 nmol/l) that block PP2A but not PP1 activity (8) did not affect the contractile responses of aortic rings from either group. Concentrations of ODA and M-LR that block both PP2A and PP1 activities (100 nmol/l for ODA and 50 nmol/l for M-LR), in contrast, enhanced maximum tension during phenylephrine-induced contraction in the hypoxia-exposed group but failed to significantly affect maximum tension in the normoxic group. Accordingly, the effect of PP1 inhibition was greater in aortic rings from hypoxia-exposed rats than in rings from normoxic rats (41.7 ± 7.4% vs. 18.2 ± 8.5% increases for ODA and 43.7 ± 1.0% vs. 13.24 ± 4.0% increases for M-LR, in hypoxic and normoxic animals, respectively; P < 0.05 for differences between groups). Incubation with the higher, but not the lower, concentration of M-LR also elevated the maximal tension elicited by KCl in aortic rings from hypoxia-exposed animals (42.9 ± 5% vs. 19.1 ± 7.4% changes in maximum tension in hypoxic and normoxic groups, respectively; P < 0.05 for difference between groups). EC50 values for phenylephrine and KCl in the presence of 100 nmol/l ODA or 50 nmol/l M-LR did not differ from those in the absence of the PP inhibitors.


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  		Fig. 1.   Concentration-response curves for phenylephrine in aortic rings from rats exposed to normoxia (A and C) and hypoxia (B and D). Rings were incubated for 20 min with Krebs-Henseleit solution (KHS; ), DMSO (open circle ), okadaic acid (black-triangle, 1 nmol/l; triangle , 100 nmol/l) or microcystin-LR (M-LR; black-down-triangle , 0.5 nmol/l; down-triangle, 50 nmol/l) before the addition of phenylephrine (n = 6 rats per group). *P < 0.05 vs. KHS and DMSO groups (ANOVA and post hoc Bonferroni's test).


                              
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  		Table 1.   Effects of phosphatase inhibitors on maximum tension and EC50 values during phenylephrine- and KCl-induced contraction

LC20 Phosphorylation

LC20 phosphorylation in aortic strips from normoxic and hypoxic rats was increased after 1 min of stimulation with 10 µmol/l phenylephrine and did not increase further after 10 min (Fig. 2). Baseline levels of LC20 phosphorylation (i.e., no stimulation) did not differ between the groups. In contrast, LC20 phosphorylation after stimulation with phenylephrine was decreased in aortas from hypoxic compared with normoxic rats (Fig. 2). M-LR (50 nmol/l) significantly elevated the proportion of phosphorylated LC20 during stimulation with phenylephrine in aortas from rats exposed to hypoxia (P < 0.05). As a result, the difference in LC20 phosphorylation during phenylephrine-induced contraction between strips from hypoxia-exposed and normoxic animals was eliminated by treatment with M-LR (Fig. 2).


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  		Fig. 2.   Phosphorylation of light chain at 20 kDa (LC20) in aortic strips from normoxic (open circle , ) and hypoxic rats (down-triangle, black-down-triangle ) after 0, 1, and 10 min stimulation with 10 µmol/l phenylephrine in the presence and absence of DMSO (open circle , down-triangle) or 50 nmol/l M-LR (, black-down-triangle ) (n = 6-7 rats for each group). The top blot is representative of samples stimulated for 10 min with phenylephrine. The bottom blot is representative of chicken gizzard myosin samples that were simultaneously extracted with the experimental samples. *P < 0.05 vs. corresponding DMSO group (ANOVA and post hoc Bonferroni's test).

Myofibrillar Phosphatase Activity Assay

Figure 3 illustrates PP1 and PP2A activities in aortic extracts from normoxic rats and rats exposed to hypoxia for 48 h. PP1 activity was greater in the myofibrillar but not the cytosolic fraction in aortas from hypoxic compared with normoxic rats (P < 0.05) whereas there was no difference in myofibrillar or cytosolic PP2A activity between the two groups.


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  		Fig. 3.   Protein phosphatase (PP) activities in cytosolic and myofibrillar extracts from normoxic and hypoxic rat aortas. PP types 1 and 2A (PP1 and PP2A) activities were evaluated as PP activity in the presence and absence of M-LR (0.5 nmol/l), respectively (n = 5 rats per group). *P < 0.05 vs. corresponding normoxic group.

Western Blots

Levels of MLCK protein in aortas from rats exposed to hypoxia for 48 h did not differ from those in the normoxic control group (Fig. 4). MYPT1 protein levels in rat thoracic aortas increased progressively with increasing duration of hypoxic exposure and these levels were not altered by removal of the endothelium (Fig. 5A). MYPT1 protein levels were also elevated in HASMCs incubated under hypoxic compared with normoxic conditions (Fig. 5B).


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  		Fig. 4.   Myosin light chain kinase (MLCK) protein levels in aortas from normoxic rats and rats exposed to hypoxia for 48 h (n = 8 rats per group). P > 0.05, differences between groups. OD, optical density.



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  		Fig. 5.   A: levels of myosin phosphatase-targeting subunit 1 (MYPT1) protein in endothelium-intact (+E) and -denuded (-E) thoracic aortas of rats exposed normoxia for 48 h and to hypoxia for 16 and 48 h; n = 5 rats per group. B: MYPT1 protein levels in human aortic smooth muscle cells cultured under normoxic and hypoxic (16 h and 48 h) conditions (n = 4 experiments per condition). *P < 0.05 vs. corresponding normoxic cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report that exposure to hypoxia is associated with reduced agonist- and depolarization-induced aortic contraction, decreased LC20 phosphorylation, and elevation of myofibrillar PP1 activity. The increase in PP1 activity, specifically localized to the contractile myofilaments, may be explained by enhanced smooth muscle cell expression of MYPT1, which was found to be increased in aortas from rats exposed to hypoxia in vivo and in cultured human aortic smooth muscle cells after hypoxic incubation.

In previous studies, the effects of prolonged exposure to hypoxia (hours to days) on in vivo pressor responses have varied depending on the experimental model. An inhibitory effect of such exposure on in vitro reactivity of systemic arteries to adrenoceptor receptor stimulation and depolarization in conduit vessels (2, 9, 37) and of myogenic responses in isolated arterioles (32), however, has been a consistent finding and indicates a widespread negative effect on systemic vascular smooth muscle contractility. In contrast to the in vivo and in vitro effects of acute hypoxia (minutes), which are predominantly mediated by the release of endothelium-derived relaxing factors (6, 27, 35) and immediately reversible on restoration of normoxia (12, 27, 35), the change in arterial smooth muscle function observed after prolonged exposure to hypoxia persists for at least 12 h after the removal of the hypoxic stimulus (2, 32), suggesting that the two responses are mediated by different mechanisms.

The contractile impairment after prolonged hypoxia is not specific for agonist-induced contraction but affects the response to membrane depolarization as well (2, 32). Moreover, neither hypoxia nor PP1 inhibition had any effect on the sensitivity (EC50) to either phenylephrine or KCl indicating that the effects of hypoxia on PP1 activity and force generation are equal across the range of agonist concentrations and membrane potentials studied and, hence, independent of events occurring at the cell membrane. Accordingly, we evaluated the effects of hypoxia on aortic levels of MLCK and MLCP, the enzymes that regulate LC20 phosphorylation and, therefore, activation of myofibrillar ATPase and cross-bridge cycling (11, 15, 17). Aortic MLCK protein levels were not decreased after hypoxic exposure, and, after PP1 inhibition with M-LR, the proportion of phosphorylated LC20 during phenylephrine-stimulated contraction was the same in aortic strips from hypoxia-exposed rats as it was in strips from normoxic rats indicating that smooth muscle activation after hypoxia is not limited by reduced MLCK activity. In contrast, we present several lines of evidence that support the hypothesis that increased myosin-targeted phosphatase activity plays an important role. Inhibition of PP1 activity increased the tension elicited by phenylephrine and KCl and the percentage of phosphorylated LC20 during phenylephrine-induced contraction to a greater degree in aortas from rats exposed to hypoxia than in aortas from normoxic animals. Myofibrillar, but not cytosolic, PP1 activity is enhanced in aortas from hypoxic rats compared with the normoxic controls. Finally, the aortic levels of MYPT1 that mediate association of PP1 catalytic activity with the myosin heavy chain are increased in the hypoxic group. These results suggest that hypoxia increases the expression of MYPT1, which localizes phosphatase activity to the thick myofilament, resulting in LC20 dephosphorylation and creating a new steady state between MLCK and MLCP activities. These findings represent the first evidence that modulation of smooth muscle cell expression of the MYPT1 subunit may play a mechanistic role in impairment of vascular smooth muscle function under pathological conditions.

Inhibition of PP1 activity with M-LR did not appreciably affect LC20 phosphorylation in aortic strips from normoxic rats (Fig. 2), and the effect on the maximum tension generated by aortic rings from normoxic animals during supramaximal stimulation with phenylephrine (10 µmol/l) also did not reach statistical significance (18.2 ± 8.5% increase compared with untreated rings, P = 0.058 for difference; see Table 1). In intact smooth muscle alpha -adrenoceptor stimulation inhibits MLCP activity by dissociating the catalytic and targeting subunits (31). Because we measured LC20 phosphorylation during maximal alpha -agonist stimulation, the extent of inhibition of myofibrillar PP1 activity necessary to induce and maintain maximum contraction must have been sufficient to preclude the detection of additional effects of exogenous inhibitors in this group. In aortic strips from rats exposed to hypoxia, PP1 inhibition has a much greater effect on both LC20 phosphorylation and contraction as would be expected if myofibrillar phosphatase activity is greatly increased and, therefore, less completely inhibited by activation of the alpha 1-receptor.

LC20 phosphorylation is the major regulatory step in the initiation of contraction, as it permits actin interaction with the myosin heavy chain. The development of force, however, depends on many other factors, some of which [e.g., modulation of Ca2+ sensitivity by telokin (34) and by thin-filament regulatory proteins (14)] are also regulated by phosphorylation and these will be affected by PP1 inhibition. Accordingly, we cannot exclude a contribution of these pathways to the increase in maximum tension elicited by PP1 inhibitors after hypoxia.

Inhibition of PP1 activity eliminated the difference in LC20 phosphorylation between normoxic and hypoxic groups in the current study; however, it did not restore the maximum tension generated by aortic rings from hypoxic rats during phenylephrine stimulation to the levels recorded in rings from normoxic animals (Fig. 6). At least one other mechanism, therefore, is concurrently responsible for the depressed reactivity. This additional abnormality is distal to myosin phosphorylation, and, therefore, represents an effect on the contractile myofilaments. Recently, we (37) evaluated contractile protein expression in aortas from rats exposed to hypoxia and normoxia for 48 h. Myosin heavy chain content was not altered after hypoxia; however, levels of the inhibitory thin filament proteins caldesmon and calponin were increased and this change in thin myofilament composition could alter the capacity for ATP hydrolysis and/or cross-bridge cycling (25). The inhibitory effects of these proteins, moreover, are Ca2+ regulated (26); therefore, changes in cellular Ca2+ handling, which was not evaluated in the present study, could also affect contractile responses through effects on myosin activation that may not be mediated by changes in LC20 phosphorylation. Interestingly, dephosphorylation of calponin, which disinhibits its negative regulatory effects, is also a PP1-mediated event in smooth muscle (14). The increase in myofibrillar PP1 activity that occurs after hypoxia may, therefore, inhibit contraction by opposing phosphorylation not only of LC20 but also, simultaneously, of calponin.


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  		Fig. 6.   Effects of PP1 inhibition with M-LR (50 nmol/l) on maximum tension in aortic rings and on LC20 phosphorylation (P-LC20) in aortic strips from normoxic rats and from rats exposed to hypoxia for 48 h during supramaximal stimulation with phenylephrine (10 µmol/l). In aortas from hypoxia-exposed rats phosphatase inhibition restored P-LC20 but not tension to the level recorded in normoxic animals. *P < 0.05 for difference between normoxic and hypoxia-exposed groups.

Total PP1 activity levels in aortas from normoxic rats in our current study were similar to those recorded previously in vascular tissues (10); however, we recovered a substantial proportion of PP1 activity (41%) in the cytosolic extracts. This contrasts with observations in chicken gizzard (1, 16) and rabbit basilar artery (10), in which 80-90% and 78% of PP1 activity, respectively, was localized to the myofibrils. Quantitative differences in myofibrillar PP1 activity and responsiveness to PP1 inhibition between tonic and phasic smooth muscles have been reported (11) and the rank order of myofibrillar PP1 activity in these studies may, therefore, reflect functional differences in the smooth muscle with that in the rat aorta being the least phasically active.

PP1 targeting subunits that bear structural similarities to smooth muscle MYPT1 have been identified in porcine and bovine endothelial cells, and a role in regulating endothelial cell contractile responses during intercellular gap formation has been proposed (13, 33). In the current study, however, removal of the endothelium had no effect on the levels of MYPT1 detected in aortas from either normoxic or hypoxia-exposed rats (Fig. 5) indicating that the increase in MYPT1 protein is localized to the medial layer and that altered endothelial cell protein expression does not contribute to the change in total aortic MYPT1 immunoreactivity in this model.

Our finding that MYPT1 levels are increased in HASMCs after hypoxic incubation suggests that the increase in MYPT1 expression is a direct effect of hypoxia on the smooth muscle rather than the result of hormonal or hemodynamic changes. The 5' flanking region of the human MYPT1 gene has been cloned previously and was found to contain functional promoter sequences (24). Analysis of this region (29) indicates the presence of consensus transcription factor elements corresponding to the aryl hydrocarbon receptor nuclear translocator required by hypoxia inducible factor-1alpha to enhance transcription of the gene encoding vascular endothelial growth factor among others (36) and for activating protein 1, which mediates hypoxic activation of tyrosine hydroxylase expression (28). Further studies are now indicated to evaluate the functionality of these putative transcription factor binding sites and their roles in the regulation of smooth muscle MYPT1 expression by oxygen tension.

Raised vascular tone in animal models of arterial vasospasm (10, 30) and vasorelaxation in response to insulin (3) and cGMP (22) have been associated, respectively, with decreased and increased myosin-targeted PP1 activity. These effects have been attributed to phosphorylation and dephosphorylation, respectively, of MYPT1 through modulation of the activity of Rho kinase rather than a change in MYPT1 levels per se. Although our current observation that aortic levels of MYPT1 are increased after hypoxia provides a mechanism for the increase in myofibrillar PP1 activity, the effect of hypoxia on Rho kinase has not been studied. Accordingly, we cannot exclude alterations in phosphorylation status of the targeting subunit as an additional mechanism contributing to inhibition of myosin phosphorylation after hypoxia, and this merits further evaluation.

The results of the present study indicate that after prolonged hypoxia MYPT1 expression and myofibrillar PP1 activity are increased in aortic vascular smooth muscle and that vasoreactivity is suppressed because of reduced LC20 phosphorylation. To the extent that the hypoxia-induced decrease in vascular smooth muscle contractility also affects arterioles that regulate flow in vital vascular beds (32), the resulting impairment of the capacity to actively regulate the systemic circulation may compromise vascular responses essential to the maintenance of arterial blood pressure and vital organ oxygenation. Because these changes occur relatively quickly (hours to days) and because hypoxia may cause organ dysfunction within this time frame during acclimatization to high altitude and during reductions in oxygen delivery due to shock and cardiopulmonary diseases, the alterations we report may contribute to the pathophysiology of these disorders.


    ACKNOWLEDGEMENTS

This study was funded by an operating grant from the Canadian Institutes of Health Research.


    FOOTNOTES

Address for reprint requests and other correspondence: M. E. Ward, Rm. 6-042, Bond Wing, St. Michael's Hospital, 30 Bond St., Toronto, Ontario, Canada M5B 1W8.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 19, 2002;10.1152/ajpheart.00680.2002

Received 1 August 2002; accepted in final form 10 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alessi, D, MacDougall LK, Sola MM, Ikebe M, and Cohen P. The control of protein phosphatase-1 by targeting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem 210: 1023-1035, 1992.

2.   Auer, G, and Ward ME. Impaired reactivity of rat aorta to phenylephrine and KCl after prolonged hypoxia: role of the endothelium. J Appl Physiol 85: 411-417, 1998.

3.   Begum, N, Duddy N, Sandu O, Reinzie J, and Ragolia L. Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. Mol Endocrinol 14: 1365-1376, 2000.

4.   Bjornheden, M, Levin M, Evaldsson M, and Wiklund O. Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol 19: 870-876, 1999.

5.   Borman, MA, MacDonald JA, Muranyi A, Hartshorne DJ, and Haystead TA. Smooth muscle myosin phosphatase-associated kinase induces Ca2+ sensitization via myosin phosphatase inhibition. J Biol Chem 277: 23441-23446, 2002.

6.   Brown, IP, Thompson CI, and Belloni FL. Role of nitric oxide in hypoxic coronary vasodilation in isolated perfused guinea pig heart. Am J Physiol Heart Circ Physiol 264: H821-H829, 1993.

7.   Cain, SM, and Chapler CK. O2 extraction by canine hindlimb during alpha -adrenergic blockade and hypoxic hypoxia. J Appl Physiol 48: 630-635, 1980.

8.   Cohen, P, Klumpp S, and Schelling DL. An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett 250: 596-600, 1989.

9.   Doyle, MP, and Walker BR. Attenuation of systemic vasoreactivity in chronically hypoxic rats. Am J Physiol Regul Integr Comp Physiol 260: R1114-R1122, 1991.

10.   Fukami, M, Tani E, Takai A, Yamaura I, and Minami N. Activity of smooth muscle phosphatases 1 and 2A in rabbit basilar artery in vasospasm. Stroke 26: 2321-2327, 1995.

11.   Gong, MC, Cohen P, Kitazawa T, Ikebe M, Masuo M, Somlyo AP, and Somlyo AV. Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle. J Biol Chem 267: 14662-14668, 1992.

12.   Heistad, DD, Wheeler RC, and Aoki VS. Reflex cardiovascular responses after 36 h of hypoxia. Am J Physiol 220: 1673-1676, 1971.

13.   Hirano, M, Niiro N, Hirano K, Nishimura J, Hartshorne DJ, and Kinaide H. Expression, subcellular localization, and cloning of the 130-kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells. Biochem Biophys Res Commun 254: 490-496, 1999.

14.   Ichikawa, K, Ito M, Okubo S, Konishi T, Nakano T, Mino T, Nakamura F, Naka M, and Tanaka T. Calponin phosphatase from smooth muscle: a possible role of type 1 protein phosphatase in smooth muscle relaxation. Biochem Biophys Res Commun 193: 827-833, 1993.

15.   Ikebe, M, Hartshorne DJ, and Elzinga M. Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-Dalton light chain of smooth muscle myosin. J Biol Chem 261: 36-39, 1996.

16.   Johnson, D, Cohen P, Chen MX, Chen YH, and Cohen PT. Identification of the regions on the M110 subunit of protein phospohatase 1M that interact with the M21 subunit and with myosin. Eur J Biochem 244: 931-939, 1997.

17.   Kamm, KE, and Stull JT. Myosin phosphorylation, force and maximal shortening velocity in neurally stimulated smooth muscle. Am J Physiol Cell Physiol 249: C238-C247, 1985.

18.   Kamm, KE, and Stull JT. Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness. Science 232: 80-82, 1986.

19.   Kimura, K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yuamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248, 1996.

20.   Kitazawa, T, Eto M, Woodsome TP, and Brautigan DL. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem 275: 9897-9900, 2000.

21.   Kuwahira, I, Gonzalez NC, Heisler N, and Piiper J. Changes in regional blood flow distribution and oxygen supply during hypoxia in conscious rats. J Appl Physiol 74: 211-214, 1993.

22.   Lee, MR, Li L, and Kitazawa T. Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase. J Biol Chem 272: 5063-5068, 1997.

23.   Lowry, OH, Rosbrough NJ, Farr AL, and Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275, 1951.

24.   Machida, H, Ito M, Okamoto R, Shiraki K, Isaka N, Hartshorne DJ, and Nakano T. Molecular cloning and analysis of the 5'-flanking region of the human MYPT1 gene. Biochim Biophys Acta 1517: 424-429, 2001.

25.   Marston, SB, Fraser ID, and Huber PA. Smooth muscle caldesmon controls the strong binding interaction between actin-tropomyosin and myosin. J Biol Chem 269: 32104-32109, 1994.

26.   Marston, SB, and Lehman W. Caldesmon is a Ca2+-regulatory component of native smooth muscle thin filaments. Biochem J 231: 517-522, 1985.

27.   Messina, EJ, Sun D, Koller A, Wolin MS, and Kaley G. Role of endothelium-derived prostaglandins in hypoxia-elicited arteriolar dilation in rat skeletal muscle. Circ Res 71: 790-796, 1992.

28.   Norris, ML, and Milhorn DE. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene. J Biol Chem 270: 23774-23779, 1995.

29.   Quandt, K, Frech K, Karas H, Wingender E, and Werner T. MatInd and MatInspector---new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23: 4878-4884, 1995.

30.   Sato, M, Tani E, Fujikawa H, and Kaibuchi K. Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res 87: 195-200, 2000.

31.   Shin, HM, Je HD, Gallant C, Tao TC, Hartshorne DJ, Ito M, and Morgan KG. Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res 90: 546-543, 2002.

32.   Toporsian, M, and Ward ME. Hyporeactivity of rat diaphragmatic arterioles following exposure to hypoxia in vivo: role of the endothelium. Am J Respir Crit Care Med 156: 1572-1578, 1997.

33.   Verin, AD, Wang P, and Garcia JG. Immunochemical characterization of myosin-specific phosphatase 1 regulatory subunits in bovine endothelium. J Cell Biochem 76: 489-498, 1999.

34.   Walker, LA, MacDonald JA, Liu X, Nakamoto RK, Haystead TA, Somlyo AV, and Somlyo AP. Site specific phosphorylation and point mutations of telokin modulate its Ca2+ sensitizing effect in smooth muscle. J Biol Chem 276: 24519-24524, 2001.

35.   Ward, ME. Dilation of rat diaphragmatic arterioles by flow and hypoxia: roles of nitric oxide and prostaglandins. J Appl Physiol 86: 1644-1650, 1999.

36.   Wiesener, MS, Munchnhagen PM, Berger I, Morgan NV, Roigas J, Schwiertz A, Jurgensen JS, Gruber G, Maxwell PH, Loning SA, Frei U, Maher ER, Grone HJ, and Eckardt KU. Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia inducible factor-1alpha in clear cell renal carcinomas. Cancer Res 61: 5215-5222, 2001.

37.   Zacour, ME, Govindaraju V, Teoh H, Halayko AJ, and Ward ME. Mechanisms of aortic smooth muscle hyporeactivity after prolonged hypoxia in rats. J Appl Physiol 92: 2625-2632, 2002.

Am J Physiol Heart Circ Physiol 284(4):H1182-H1189
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